Recombinant Chicken Protein NEF1 (NEF1)

What is chicken NAIF1 and what are its key biological functions?

Chicken NAIF1 (Nuclear Apoptosis Inducing Factor 1) is encoded by gene ID 417229 in Gallus gallus, with the UniProt ID Q5ZI27 and protein reference sequence NP_001025863 . As its name suggests, NAIF1 functions primarily as a nuclear apoptosis-inducing factor, playing a crucial role in programmed cell death pathways. The protein is involved in cellular stress response mechanisms and may contribute to tissue homeostasis in avian systems. Unlike some other recombinant proteins that have been extensively studied in viral vector systems (such as HIV-1 Nef proteins in influenza vectors), the precise signaling pathways and molecular interactions of chicken NAIF1 require further characterization in current research .

How does recombinant chicken NAIF1 differ from its naturally occurring counterpart?

Recombinant chicken NAIF1 is produced in expression systems (typically mammalian cells for optimal post-translational modifications) and often includes fusion tags for purification and detection purposes. The commercially available recombinant chicken NAIF1 is typically produced with a His-tag and has a purity of >80% as determined by standardized methods . The recombinant version maintains the functional domains of the native protein but may have slight differences in folding or activity depending on the expression system used. When designing experiments, researchers should consider that the presence of purification tags may potentially influence protein-protein interactions or enzymatic activity in some experimental contexts.

What expression systems are most effective for producing recombinant chicken NAIF1?

Based on commercial production practices, mammalian cell expression systems are preferred for recombinant chicken NAIF1 production to ensure proper folding and post-translational modifications . While bacterial systems might offer higher yields, they often fail to reproduce the correct disulfide bonding patterns and post-translational modifications that may be essential for NAIF1 function. For research requiring higher purity or specific tagging configurations, custom production services offer mammalian cell-based expression with various fusion tags (including His, GST, or Fc tags) with production lead times of 5-9 weeks . When selecting an expression system, researchers should consider:

What are the optimal storage and handling conditions for maintaining recombinant chicken NAIF1 stability?

Recombinant chicken NAIF1 is typically supplied either as a liquid formulation in PBS buffer or as a lyophilized powder. For short-term storage (up to 1 month), the protein can be stored at +4°C. For long-term preservation of activity, storage at -20°C to -80°C is recommended . When working with the protein, minimize freeze-thaw cycles as they can lead to protein denaturation and loss of biological activity. If multiple uses are anticipated, aliquoting the protein before freezing is advised. For optimal results, researchers should verify protein stability using activity assays specific to their experimental system after prolonged storage periods.

How can recombinant chicken NAIF1 be used to study apoptotic pathways in avian cell models?

Recombinant chicken NAIF1 can serve as a valuable tool for investigating apoptotic pathways specific to avian systems. Researchers can use purified recombinant NAIF1 for:

• Overexpression studies: Transfection of chicken cell lines (such as DF1 cells) with expression vectors containing chicken NAIF1 to study dose-dependent effects on apoptosis induction .

• Interaction studies: Using recombinant NAIF1 in pull-down assays to identify binding partners within apoptotic pathways.

• Localization studies: Fluorescently tagged NAIF1 can be used to monitor subcellular localization during apoptosis progression.

• Comparative studies: Researchers can compare the effects of NAIF1 with other apoptosis-inducing factors to establish pathway hierarchies and crosstalks.

When designing these experiments, it's important to include appropriate controls, such as cells expressing inactive NAIF1 mutants or non-relevant nuclear proteins of similar size and charge characteristics.

How does chicken NAIF1 compare structurally and functionally to its mammalian homologs?

Comparative analysis between chicken NAIF1 and its mammalian counterparts reveals both conservation and divergence. Researchers investigating these differences should consider:

• Sequence alignment analysis to identify conserved domains and species-specific regions.

• Structural prediction and modeling to compare folding patterns and potential functional domains.

• Functional complementation assays to determine if chicken NAIF1 can rescue phenotypes in mammalian cells with NAIF1 knockdown.

The search results indicate that recombinant NAIF1 from various species (including human, rhesus macaque, and chicken) is commercially available , facilitating direct comparative studies. These comparisons can provide insights into the evolutionary conservation of apoptotic pathways and species-specific adaptations.

What are common challenges in purifying functional recombinant chicken NAIF1 and how can they be addressed?

Researchers working with recombinant chicken NAIF1 may encounter several technical challenges:

• Protein solubility issues: NAIF1 may form inclusion bodies in some expression systems. Solution: Optimize expression conditions (temperature, induction parameters) or use solubility tags (SUMO, MBP).

• Purity limitations: Standard purification may achieve >80% purity , which may be insufficient for certain applications. Solution: Implement multi-step purification strategies combining affinity chromatography with size exclusion or ion exchange methods.

• Endotoxin contamination: Expression in bacterial systems can introduce endotoxins. Solution: Use endotoxin removal techniques and verify levels using LAL assays (standard threshold: <1.0 eu per μg of protein) .

• Protein activity loss: Storage or purification conditions may affect functional activity. Solution: Include activity assays at each purification step and optimize buffer conditions for stability.

How can researchers verify the functional activity of purified recombinant chicken NAIF1?

Verifying functional activity is crucial before using recombinant chicken NAIF1 in experiments. Recommended approaches include:

• Apoptosis induction assay: Treating chicken cell lines with purified NAIF1 and measuring apoptotic markers (caspase activation, phosphatidylserine externalization, DNA fragmentation).

• Nuclear localization verification: Using immunofluorescence to confirm proper nuclear targeting of the recombinant protein.

• Binding partner validation: Confirming interaction with known NAIF1 binding partners using pull-down or co-immunoprecipitation assays.

• Comparative activity assay: Benchmarking activity against commercially available standards when possible.

For recombinant proteins used in functional studies, it's advisable to include both positive controls (known active forms) and negative controls (heat-inactivated protein or irrelevant proteins) to establish specificity of observed effects.

How can recombinant chicken NAIF1 research be integrated with studies on avian immune responses?

Integrating NAIF1 research with broader avian immunology studies could reveal important connections between apoptotic pathways and immune function. Potential approaches include:

• Examining NAIF1 expression and activity in various immunological contexts (viral infection, immune cell activation).

• Investigating potential interactions between NAIF1 and immune signaling pathways.

• Exploring whether NAIF1 influences immune cell development or function through selective apoptosis.

Studies on viral interactions with chicken proteins have demonstrated important immunomodulatory effects. For example, research on Newcastle disease virus has identified specific chicken proteases (Tmprss9, Tmprss4, F7, Cfd, and Prss23) that activate the virus . Similar methodological approaches could be applied to investigate if NAIF1 interacts with viral components or influences interferon response pathways during infection, similar to how chicken MERTK protein has been shown to suppress Newcastle disease virus replication through the IFITM3 pathway and enhance STAT1 phosphorylation in type I IFN signaling .

What considerations are important when designing gene-editing experiments targeting chicken NAIF1?

Researchers planning CRISPR-Cas9 or other gene-editing approaches to modify chicken NAIF1 should consider:

• Guide RNA design: Target unique regions of the NAIF1 gene (ID: 417229) with minimal off-target potential in the chicken genome.

• Cellular models: DF1 cells have been successfully used for chicken protein studies and viral interactions , making them suitable for initial NAIF1 gene editing experiments.

• Phenotypic validation: Establish clear readouts for NAIF1 function disruption, potentially including apoptosis resistance, altered nuclear morphology, or disrupted protein-protein interactions.

• Genetic compensation: Monitor for potential upregulation of functionally redundant genes that might mask phenotypes.

• Rescue experiments: Include complementation with wild-type NAIF1 to verify specificity of observed phenotypes.

What emerging technologies could advance our understanding of chicken NAIF1 function?

Several cutting-edge approaches could significantly enhance our understanding of chicken NAIF1:

• Cryo-EM structural analysis: Determining the high-resolution structure of chicken NAIF1 to inform functional studies and comparative analyses with mammalian homologs.

• Proximity labeling approaches (BioID, APEX): Identifying the dynamic interactome of NAIF1 in different cellular contexts.

• Single-cell analyses: Examining cell-to-cell variation in NAIF1 expression and its correlation with cellular phenotypes in heterogeneous tissues.

• Viral vector systems: Adapting approaches used with other recombinant proteins, such as the influenza virus vectors used for HIV-1 Nef protein expression , to study NAIF1 in various contexts.

How might recombinant chicken NAIF1 contribute to comparative studies across species?

Comparative studies using recombinant NAIF1 from different species could reveal evolutionary adaptations in apoptotic pathways:

• Cross-species complementation: Testing if chicken NAIF1 can functionally replace its counterparts in mammalian cells and vice versa.

• Structural comparisons: Identifying conserved and divergent domains through comparative structural biology.

• Interaction profile analysis: Determining if NAIF1 interacts with the same partner proteins across species.

• Species-specific regulatory mechanisms: Investigating differences in post-translational modifications or localization patterns.

The availability of recombinant NAIF1 from multiple species, including human, rhesus macaque, and chicken , provides resources for such comparative studies.